1. Field of the Invention
The present invention relates to the field of vaccine production and preventing or treating infectious diseases. Specifically, the present invention relates to methods for producing a safe and effective vaccine and methods for enhancing an effective immune response in a host animals subsequently exposed to infection by bacterial pathogens, for example, Mycobacterium tuberculosis. 
2. Background Art
Although recombinant DNA technology promises the ability to make a new generation of rationally designed live-attenuated vaccines, there are critical problems that have limited the usefulness of traditional allelic inactivation techniques in fulfilling this promise.
A major problem common to all vaccine development including those created by using recombinant DNA technology has been the difficulty in inducing responses in specific populations of immune cells capable of rendering long-lived protection. Vaccination prepares an animal to respond to antigens of pathogenic microbes. Vaccination is more complex than immune recognition in that only some types of immune responses will be correlated with protective immunity and the responses that are important vary with different pathogens. For example, antibody responses are critical in conferring protective immunity against polio but have minimal importance in immune protection against tuberculosis.
With many pathogens a cellular immune response is more important than an antibody response. Cellular immune responses are primarily mediated by CD4+ T-lymphocytes, which secrete interferon-gamma and activate macrophages to kill intracellular microbes, and by CD8+ T-lymphocytes, which induce cytotoxic responses via death receptors (e.g., Fas, TNF-alpha receptor) or granular enzymes (i.e., perforin/granzyme), in addition to secreting interferon-gamma.
CD4+ lymphocytes and CD8+ lymphocytes are primed for an immune response using different antigen presentation pathways. With CD4+ T-cells, exogenous foreign antigens are taken up or recovered from ingested microbes within the phagosome of antigen presenting cells, degraded into fragments, and bound on the surface of these cells to MHC Class II molecules for presentation to the CD4+ T-cells. MHC Class II molecules are restricted primarily to some few types of lymphoid cells. CD8+ T-cell activation is achieved via a different mechanism that involves MHC Class I molecules, which are found on essentially all nucleated cells. Proteins produced or introduced within the nucleated cell are degraded to peptides and presented on the cell surface in the context of MHC Class I molecules to CD8+ T-cells. MHC Class I antigen presentation is generally referred to as the “endogenous antigen” pathway to differentiate it from the “exogenous antigen” pathway for presenting antigens via MHC Class II receptors to CD4+ T-lymphocytes.
Dendritic cells are specialized lymphoid cells that are especially efficient in presenting antigens. They have the capability of internalizing apoptotic macrophages or their fragments and presenting antigens from within the apoptotic macrophage via both MHC Class I and MHC Class II pathways (Albert, Sauter, and Bhardwaj, 1998; Yrlid and Wick, 2000; Wick and Ljunggren, 1999).
With many infectious diseases CD8+ T-cells appear to be the major determinant of prolonged immunity against re-infection that does not depend upon persistent antigenic stimulation (Lau et al., 1994). This has made the generation of strong CD8+ responses a critical factor for new vaccines. Some strategies such as DNA vaccination (Feigner et al., U.S. Pat. No. 5,589,466) appear to produce relatively good CD8+ responses compared to other methods of vaccination. However, a significant limitation of DNA vaccination is that antigens are derived from only a single gene or only a small portion of a bacterial genome corresponding to a minority of the microbe's immunodominant CD8+ epitopes. A method for delivering a larger repertoire of microbial antigens to dendritic cells would be useful, including techniques for directing whole-cell live-attenuated vaccines to associate with dendritic cells so that all microbial antigens could be processed.
Aside from the general problem common to all vaccines of inducing the appropriate type of protective immune responses for a specific pathogen, there are also specific problems with using allelic inactivation to construct new live-attenuated vaccines. One is that many microbial genes are essential for survival, including some of the best potential targets for attenuating a virulent microbe to produce a new vaccine candidate. Therefore, a mutant wherein the activity of a gene that is essential for in vitro growth has been inactivated cannot be cultivated in vitro. This problem can be partially overcome if exogenous substances are added to bypass the function of the essential gene, for example, supplementing in vitro culture media with amino acids or hypoxanthine to permit the identification and recovery of allelic knockout mutants in which essential genes for amino acid biosynthesis or purine biosynthesis, respectively, have been inactivated. Another strategy to overcome this limitation has been “in vivo expression technology” and other techniques for identifying differentially expressed genes (Slauch, Mahan, and Mekalanos, 1994). These techniques attempt to identify genes that are essential for the in vivo survival of a microbe but which are not essential for their in vitro cultivation. Therefore, it is anticipated that inactivating the “in vivo”-expressed gene will yield a candidate that can be cultivated in vitro but is attenuated in vivo.
However, although the exogenous supplementation of auxotrophs and the identification of genes that are essential only for in vivo microbial survival has expanded the number of genes that can be inactivated to generate new live-attenuated vaccine candidates, a large number of genes remain that are essential both for in vitro and in vivo microbial survival, and which are not amenable to exogenous supplementation to enable their in vitro survival and cultivation. Therefore, having a method that enables a partial rather than complete reduction in the gene product will permit a larger number of such essential genes to be altered for producing new live-attenuated vaccine candidates. Ideally, this method would enable the production of stable mutants with minimal potential to revert back to the virulent phenotype.
A second major problem with current attempts to create new vaccines using recombinant DNA technology has been the difficulty in finding the right balance between attenuation and immunogenicity. In most circumstances when a mutant is identified or created, it is either still too virulent to be used as a vaccine or is cleared too rapidly by the host to engender protective immunity. For example, new auxotrophic tuberculosis vaccine candidates exemplify this problem all too well. If the strain can scavenge too much of the essential nutrient in vivo, it may be able to assume the virulent phenotype. Consider that methionine-auxotrophic mutants of Bacillus Calmette-Guerin (BCG), a vaccine strain for tuberculosis that is given to more than 100 million people annually worldwide, grow as well in vivo as the parent BCG strain (McAdam et al., 1995). This suggests that the auxotroph is able to scavenge enough methionine in vivo to meet its growth needs, thereby bypassing the metabolic defect that made it auxotrophic in vitro. This makes it likely that a methionine-auxotrophic mutant of virulent M. tuberculosis would still be virulent in vivo. In contrast, if growth is restricted too severely the auxotroph might not persist long enough or make enough of the immunodominant antigens needed to drive a protective immune response. This has been observed with a lysine auxotroph, which when given as a single-dose immunization had no efficacy, but conferred some protective immunity when given as a 3-dose regimen (Collins et al., 2000). The leucine and purine auxotrophs have shown some vaccine efficacy but appear less protective than immunization with BCG (Hondalus et al., 2000; Jackson et al., 1999).
A further problem with current allelic inactivation strategies for vaccine development is the inability to subsequently modify a promising vaccine candidate. When allelic inactivation creates a mutant that is too attenuated, then it is not possible using allelic inactivation techniques for the mutant to be further adjusted to achieve the right balance between attenuation and immunogenicity. When allelic inactivation creates a mutant that is not sufficiently attenuated, then allelic inactivation can be applied to a second microbial gene in an effort to further attenuate the mutant, but there still remains a difficulty in obtaining the optimal level of attenuation for vaccine efficacy. Therefore, allelic inactivation is an unpredictable tool for fine-tuning the level of attenuation. However, achieving the right balance between attenuation and immunogenicity is recognized as being critical to the development of a new live-attenuated tuberculosis vaccine, and strategies that enable incremental adjustments of vaccine candidates are needed.
Historically one of mankind's most important infections, tuberculosis remains a major public health problem and cause of human illness. Morbidity and mortality remain high with predictions of 225 million new cases and 79 million deaths from tuberculosis between 1998 and 2030 (Murray and Salomon, 1998). This is despite the availability of effective treatment regimens and the widespread use of a live attenuated strain of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG), as a vaccine. Furthermore, there is an enormous reservoir of persons latently infected with Mycobacterium tuberculosis estimated to comprise approximately one-third of the world's population (Snider, Jr. and La Montagne, 1994).
Previous investigations have shown that BCG does not induce very strong CD8+ responses (Kaufmann, 2000). However, MHC Class I pathways and CD8+ T-cells are important in the normal host containment of virulent M. tuberculosis, as mice with defects in MHC Class I antigen presentation succumb rapidly to tuberculosis (Flynn et al., 1992;Sousa et al., 2000). MHC Class I pathways and CD8+ T-cells may be particularly important in preventing latent tuberculosis infection from developing into active disease, as exposed healthy household contacts of TB cases and individuals with inactive self-healed TB exhibit vigorous CD8+ responses (Lalvani et al., 1998) (Pathan et al., 2000). CD8+ cells appear to exert their protective effect against tuberculosis in several ways including cytotoxic T-lymphocyte (CTL) activity resulting in lysis of host macrophages infected with M. tuberculosis, by killing intracellular bacilli via the release of the antimicrobial peptide granulysin, and by IFN-gamma production (Cho et al., 2000; Serbina and Flynn, 1999; Silva et al., 1999; Serbina et al., 2000). As classic concepts regarding host control of tuberculosis have emphasized the processing of antigens from M. tuberculosis within phagosomes via MHC Class II pathways to CD4+ T-cells, it has been unclear why MHC Class I-CD8+ pathways should be so critical.
Devising strategies to induce greater CD8+ responses than BCG is a major focus of tuberculosis vaccine research (Cho, Mehra, Thoma-Uszynski, Stenger, Serbina, Mazzaccaro, Flynn, Barnes, Southwood, Celis, Bloom, Modlin, and Sette, 2000; Kaufmann, 2000). Such appears to be possible via DNA vaccination, which results in greater populations of CD8+/CD44hi T-cells that produce IFN-gamma than is achieved with BCG vaccination (Silva, Bonato, Lima, Faccioli, and Leao, 1999). However the T-cell responses are restricted to the antigen or antigens produced by the specific gene or genes expressed by the DNA vaccine. Others have tried to improve CD8+ responses by cloning listeriolysin into BCG in hopes that its pore-forming properties would enable M. tuberculosis antigens within the phagosome to gain access to the cytosol of the macrophage and be processed via MHC Class I pathways (Hess et al., 1998).
Because of the inability of the BCG vaccine to adequately prevent pulmonary infection by M. tuberculosis, there exists a need to improve antigen presentation to the immune cells most strongly correlated with long-term protective immunity, particularly CD8+ T-lymphocytes. The present invention provides a solution to this problem by providing a method of vaccine development, wherein the pathogen is engineered so that antigen presentation is enhanced.
There also exists a great need for developing a vaccine that is derived from a bacterium that retains sufficient immunogenicity and can be modified to attenuate its pathogenicity. Accordingly, there exists a need for a method of vaccine development that provides the right balance between attenuation and immunogenicity. The present invention provides the solution to this problem by providing a method of vaccine development, wherein the pathogen is engineered so that an enzyme essential for the full expression of survival in vivo is produced at reduced levels or is a less-efficient form of the enzyme, yet the live-attenuated microbe is sufficiently immunogenic.
The invention specifically solves the problems with M. tuberculosis vaccines by providing an effective vaccine against tuberculosis.